DNA-binding proteins are important biomolecules for life. Interactions of DNA-binding proteins with DNA at sequence-specific sites regulate gene expression during particular cellular events. Interactions between DNA-binding proteins and their target sequences in DNA are essential for many crucial cellular processes such as, for example, gene transcription, gene replication and gene recombination. DNA-binding proteins may be involved in regulation of various cellular mechanisms including, for example, control of cell cycle, cell death, cell response to various external signaling events, and cell metabolism.
The biological functions regulated by DNA-binding proteins (e.g., transcription factors) underlie their importance as biomarkers or targets for disease diagnosis and/or drug development. Thus, it is desirable to develop simple and robust methods for identifying DNA-binding proteins and to monitor their DNA-binding activities. Preferably, such identification methods would be amenable to high-throughput screening techniques.
To date, methods to detect DNA-binding proteins with sequence specificity such as, for example, fluorescent titration methods, gel shift assays, and DNA foot-printing assays are complicated, labor-intensive and time-consuming. The use of radioactive or fluorescent labels and specialized instruments/facilities in most of these techniques imposes limitations on cost, safety, usability and sensitivity for practical use. Such techniques are not adaptable to high-throughput formats, as often needed in biomedical research.
Metal nanoparticles (mNP) have unique optical properties which arise from their ability to support a localized surface plasmon resonance (LSPR). As a result of these unique optical properties, a solution of nanoparticles has a characteristic color, which can change depending on changes in the nanoparticles and/or the arrangement of the nanoparticles. Bioassays using nanoparticles have been developed for a wide range of analytes such as, for example, DNA, metal ions, and small molecules.
One straightforward method for mNP-based colorimetric assays involves functionalizing two sets of mNPs separately, each with probe and target biomolecules, and then directly detecting aggregation of the differently functionalized mNPs as a result of direct recognition between probe and target. This strategy is less than ideal for the design of protein-related assays because conjugation may change the conformation of a protein and affect the specific biomolecular interactions in which the protein is involved.
Many metal nanoparticles (mNP)-based colorimetric assays for DNA binding proteins are designed based on a crosslinking mechanism, in which two sets of mNP-conjugates are linked/assembled (in the presence of analyte) through permanent interparticle bond formation. This on-particle biorecognition process is slow and can take up to 12 hours to observe a color change. In addition, such analyte-induced aggregation mechanisms tend to produce false positive results caused by destabilizing effects of unrelated molecules that may be present in the reaction buffer, causing aggregation of the mNPs.
In contrast to a crosslinking mechanism, non-crosslinking mechanisms use unmodified mNPs that involve no inter-particle linkage and may provided relatively fast colorimetric response (within minutes). The stability of the mNPs is achieved via the controlled loss and/or gain of stabilization forces on the particle surface. For example, the addition of a salt to the reaction buffer may neutralize surface charges on the mNPs, causing the mNPs to aggregate. However, this type of assay is not suitable for use in protein sensing due to the largely available non-specific binding of proteins to the bare mNPs surface.
Other strategies involve the use of particle aggregates to detect a protein analyte that dissociates the aggregates into dispersed particles. A typical example is the endonuclease (DNase I) sensor for the detection of enzymatic cleavage activity and inhibition. This technology involves intensive inter-particle hybridization and requires careful monitoring of melting (dissociation) behavior of DNA. In addition, due to inaccessibility of DNA embedded inside the particle aggregates, this assay is limited in application, and is not useful for proteins that are large in size or which do not possess an enzymatic cleavage function.